CN102220228A - Polymerase chain reactor and real-time optical array detection device - Google Patents
Polymerase chain reactor and real-time optical array detection device Download PDFInfo
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- CN102220228A CN102220228A CN2011101340454A CN201110134045A CN102220228A CN 102220228 A CN102220228 A CN 102220228A CN 2011101340454 A CN2011101340454 A CN 2011101340454A CN 201110134045 A CN201110134045 A CN 201110134045A CN 102220228 A CN102220228 A CN 102220228A
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Abstract
The invention relates to a polymerase chain reactor and a real-time optical array detection device, which are applied in the field of biochemical reaction and medical detection. The polymerase chain reactor comprises a base layer with a spiral microfluidic channel structure, wherein the base layer is sealed with a cover board layer through bonding technology, the cover board layer is provided with two through holes including a sample incoming hole and a sample outgoing hole, and a microprocessor is adopted to control three semi-conductor heating and refrigerating devices, so that a whole microfluidic channel area forms three constant temperature areas, namely a degeneration area, an annealing area and an extension area needed by polymerase chain reaction; and three platinum resistance temperature sensors are arranged among a microfluidic chip and the two semi-conductor heating and refrigerating devices, so that a closed ring is formed to control temperature. The real-time optical array detection system comprises a light source, a photoelectric detector and a fluorescence signal acquisition processing system. In the invention, as the microfluidic chip is made by MEMS (Micro-electromechanical System) technology, a reagent is miniaturized; and as the three constant temperature areas are designed, the temperature rising and reducing time is saved, the time for polymerase chain reaction is shortened, and the service life of the semi-conductor heating and refrigerating device is prolonged.
Description
Technical field
(Polymerase Chain Reaction PCR) realizes that on micro-fluidic chip DNA cloning and real-time fluorescence detect to the present invention relates to the polymerase chain reaction.Be applied to biochemical reaction and medical science detection range.
Background technology
Microminiaturization is a developing direction in the biochemistry detection apparatus field, and (Micro-electro-mechanical systems, development MEMS) can be finished the PCR reaction to micro-electromechanical technology on chip.Have following shortcoming in the conventional fluorescent PCR instrument: the instrument volume is big, and temperature rate is slow, and temperature accuracy is not high, the long-term temperature shock lost of life of semi-conductor heating cooler.The little well formula chip that utilizes the MEMS technology to make solved the bulky problem of instrument, but above-mentioned other problems exists also.In three flat-temperature zone micro-fluidic chips, can address the above problem, but because the difficult problem that the polymerase chain reaction real-time fluorescence detects on liquid-flow dynamical problem and the realization micro-fluidic chip has appearred again in constructional feature.
Summary of the invention
The object of the invention provides a kind of miniature polymerase chain reaction device and real-time optical array detection device that can carry out fluoroscopic examination simultaneously in the reaction of the enterprising performing PCR of micro-fluidic chip, and it has accuracy of temperature control height, characteristics that stability is strong.
For achieving the above object, the present invention takes following technical scheme: polymerase chain reaction device and real-time optical array detection device comprise:
Micro-fluidic chip, this system has stratum basale 1, the cover layer 2 of volution fluid channel structure, and stratum basale 1 forms micro-fluidic chip with cover layer 2; On cover layer 2, there are two holes to be respectively sample holes 3 and sample outlet hole 4, lay respectively at the starting point and the terminal point of fluid channel; The pneumatic pump that provides pneumatic power to make sample flow is connected to sample holes 3; Stratum basale 1 material is the good material of heat conduction; Cover layer 2 is a light transmissive material;
The thermal cycling unit, this system comprises semi-conductor heating cooler 5 and driving circuit, temperature sensor 6, and based on the control module of microprocessor and proportion integration differentiation algorithm; 3 semi-conductor heating cooler 5 constitute sex change, annealing, three districts of extension below annular microfluidic channel; Temperature sensor 6 is placed between semi-conductor heating cooler 5 and the micro-fluidic chip; Temperature sensor 6 is connected to control module, and control module is connected to driving circuit, and driving circuit links to each other with semi-conductor heating cooler 5, the heating or the refrigeration temperature of control semi-conductor heating cooler 5;
Real-time fluorescence detecting unit, this unit comprise light source 8, photodetector 9, reach above-mentioned control module; Light source 8 is adopted the linear array photodiode, and photodetector 9 adopts the linear array avalanche photodides, and linear array LED and linear array avalanche photodide be arranged in parallel, and becomes angle greater than 10 ° angle with spiral fluid channel, and is fixing by optics support 7; Linear array LED and linear array avalanche photodide are placed on micro-fluidic chip extension area top; Control module is connected to photodetector 9.
Control module in above-mentioned thermal cycling unit and the real-time fluorescence detecting unit is connected to computer.
Described stratum basale 1 and cover layer 2 are with the bonding techniques formation micro-fluidic chip that is sealed.
Described temperature sensor 6 is a platinum sensor; Semi-conductor heating cooler 5, temperature sensor 6, light source 8, employing semiconductor technology are made.
Described control module is to be the control core module to adopt digital signal processor DSP or on-site programmable gate array FPGA.
The computer interface of controlling whole device adopts serial ports or USB or bluetooth or Wi-Fi or the wired or wireless mode of internet network to be connected to control module.
The representative temperature in described sex change district is 95 ℃, and the representative temperature of annealed zone is 65 ℃, and the representative temperature of extension area is 72 ℃.
Working process of the present invention is as follows, sample flows into fluid channel by sample holes, sample flows in fluid channel under the driving of pneumatic pump, sample is a mobile circle in fluid channel, sex change district, annealed zone, extension area have promptly been flow through respectively, finish the polymerase chain reaction one time, the linear array led lighting once, isochrone snow shower avalanche photo diode carries out first order fluorescence to reaction and surveys; Circulating repeatedly and realize the amplification of DNA in the polymerase chain reaction, carries out real-time fluorescence simultaneously and detect.
The present invention has substantial characteristics and progress, technique effect of the present invention: compare with the PCR in real time instrument of classics, utilize MEMS fabrication techniques micro-fluidic chip, the reagent traceization; Save the heating and cooling time by the design of three flat-temperature zones and shortened the time of polymerase chain reaction greatly, prolonged the work-ing life of semi-conductor heating cooler simultaneously.Compare with the PCR micro-fluidic chip of having invented, on chip, make the semi-conductor heating cooler and replace existing micro-heater, not only can heat but also can active refrigeration; Invent a highly sensitive real-time fluorescence detection system of low cost, the linear array photodiode that light source adopts semiconductor technology to make, photodetector adopts the linear array avalanche photodide, realizes that real-time quantitative fluorescence detects.Owing to adopt the volution fluid channel, make sample easier to be mobile in runner.When detecting, only need to change micro-fluidic chip, reduced experimental cost for a plurality of samples.
Description of drawings
Below in conjunction with accompanying drawing patent of the present invention is further specified.
Fig. 1 structure vertical view of the present invention.
Fig. 2 structure side-looking of the present invention side.
Fig. 3 is the A-A sectional view of Fig. 1.
Fig. 4 system chart of the present invention.
Among the figure 1, have a stratum basale of volution fluid channel, 2, cover layer, 3, sample holes, 4, sample outlet hole, 5, the semi-conductor heating cooler, 6, temperature sensor, 7, the optics support, 8, light source, 9, photodetector, 10, pneumatic pump.
Embodiment
Be described further for the present invention below in conjunction with the drawings and specific embodiments:
As illustrated in fig. 1 and 2, polymerase chain reaction of the present invention device and real-time optical array detection device comprise micro-fluidic chip, pneumatic pump, temperature controlling system and fluorescence detection device.Being temperature controlling system below fluid channel, is fluorescence detection device above fluid channel, and pneumatic pump is not done concrete regulation as long as realize its position of function.As shown in Figure 3, be cover layer 2 successively from top to bottom, have stratum basale 1, temperature sensor 6 and a semi-conductor heating and cooling sheet 5 of volution fluid channel.
The micro-fluidic chip mode of connection utilizes the manufacturing of MEMS technology to have the stratum basale 1 and the cover layer 2 of volution fluid channel, and both become micro-fluidic chip by bonding.On cover layer 2, there are two communicating poress to be respectively sample holes 3, sample outlet hole 4, sample holes 3, sample outlet hole 4 are corresponding with the starting point and the terminal point of fluid channel respectively, sample is from sample holes 3 sample introductions, pneumatic pump 10 is from sample holes 3 air inlets behind the sample introduction, sample flows by the air pressure mode, and unnecessary gas is discharged from sample outlet hole 4.The flow velocity of sample can be controlled by the air input of pneumatic pump 10, and then the real time position of sample can be known according to fluid channel size and sample flow rate.Concrete regulation is not done in the position of pneumatic pump 10, regulates as the case may be, as long as realize its function.
Be three semi-conductor heating cooler 5 below micro-fluidic chip, it covers whole fluid channel zone, leaves certain space between three semi-conductor heating cooler 5, utilizes air heat insulation.Make fluid channel sex change district constant temperature 95 ℃ of representative temperatures, for denaturation process provides constant temperature by control sex change district semi-conductor heating cooler 5; Make fluid channel annealed zone constant temperature 65 ℃ of representative temperatures, for annealing process provides constant temperature by control annealed zone semi-conductor heating cooler 5; Make fluid channel extension area constant temperature 72 ℃ of representative temperatures by control extension area semi-conductor heating cooler 5, for the extension process provides constant temperature.1 minute sex change stage, annealing stage 30 seconds, the extension stage is then decided according to the dna fragmentation length that will increase, and generally needs 1 minute.Sample is finished the polymerase chain reaction one time through sex change, annealing and extension, and the polymerase chain reaction process circulates repeatedly and realizes the amplification of DNA.Before the amplified reaction, need pre-sex change in 10 minutes usually.
Be the real-time fluorescence proofing unit above micro-fluidic chip, comprise light source, photodetector 9 and fluorescent signal acquisition processing system.The linear array photodiode that light source 8 adopts semiconductor technology to make, photodetector 9 adopts the linear array avalanche photodide, linear array LED and linear array avalanche photodide be arranged in parallel, and become greater than 10 ° of angles with spiral fluid channel, fixing by optics support 7, be placed on micro-fluidic chip extension area top, sample is a mobile circle in fluid channel, sex change district, annealed zone, extension area have promptly been flow through respectively, finish the polymerase chain reaction one time, the linear array led lighting once, isochrone snow shower avalanche photo diode carries out first order fluorescence to reaction and surveys; Circulating repeatedly and realize the amplification of DNA in the polymerase chain reaction, carries out real-time fluorescence simultaneously and detect.The controlled module controls of fluorescent signal acquisition processing system and temperature controlling system, control module links to each other with computer, as shown in Figure 4, computer interface adopts serial ports or USB or bluetooth or wired or wireless modes such as Wi-Fi or internet network, and host computer procedure is controlled whole device.Control module is to be the control core module to adopt digital signal processor DSP or on-site programmable gate array FPGA.
Claims (6)
1. polymerase chain reaction device and real-time optical array detection device is characterized in that it comprises:
Micro-fluidic chip, this system has stratum basale (1), the cover layer (2) of volution fluid channel structure, and stratum basale (1) forms micro-fluidic chip with cover layer (2); On cover layer (2), there are two holes to be respectively sample holes (3) and sample outlet hole (4), lay respectively at the starting point and the terminal point of fluid channel; The pneumatic pump that provides pneumatic power to make sample flow is connected to sample holes (3); Stratum basale (1) material is the good material of heat conduction; Cover layer (2) is a light transmissive material;
The thermal cycling unit, this system comprises semi-conductor heating cooler (5) and driving circuit, temperature sensor (6), and based on the control module of microprocessor and proportion integration differentiation algorithm; 3 semi-conductor heating cooler (5) sample flow direction below annular microfluidic channel constitutes sex change, annealing, three districts of extension successively; Temperature sensor (6) is placed between semi-conductor heating cooler (5) and the micro-fluidic chip; Temperature sensor (6) is connected to control module, and control module is connected to driving circuit, and driving circuit links to each other with semi-conductor heating cooler (5), the heating or the refrigeration temperature of control semi-conductor heating cooler (5);
Real-time fluorescence detecting unit, this unit comprise light source (8), photodetector (9), reach above-mentioned control module; Light source (8) is adopted the linear array photodiode, and photodetector (9) adopts the linear array avalanche photodide, and linear array LED and linear array avalanche photodide be arranged in parallel, and becomes angle greater than 10 ° angle with spiral fluid channel, and is fixing by optics support (7); Linear array LED and linear array avalanche photodide are placed on micro-fluidic chip extension area top; Photodetector (9) is connected to control module;
Above-mentioned control module is connected to computer.
2. polymerase chain reaction according to claim 1 device and real-time optical array detection device is characterized in that: described stratum basale (1) and cover layer (2) are with the bonding techniques formation micro-fluidic chip that is sealed.
3. polymerase chain reaction according to claim 1 device and real-time optical array detection device is characterized in that: described temperature sensor (6) is a platinum sensor; Semi-conductor heating cooler (5), temperature sensor (6), light source (8) adopt semiconductor technology to make.
4. polymerase chain reaction according to claim 1 device and real-time optical array detection device is characterized in that: described control module is to be the control core module to adopt digital signal processor DSP or on-site programmable gate array FPGA.
5. polymerase chain reaction according to claim 1 device and real-time optical array detection device is characterized in that: the computer interface of controlling whole device adopts serial ports or USB or bluetooth or Wi-Fi or the wired or wireless mode of internet network to be connected to control module.
6. polymerase chain reaction according to claim 1 device and real-time optical array detection device is characterized in that: the described semi-conductor heating cooler of reversal connection (5) can realize the refrigeration cooling of three warm areas, has replaced traditional naturally cooling.
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Cited By (7)
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| CN105296351A (en) * | 2015-12-02 | 2016-02-03 | 北京大学 | Chip for polymerase chain reaction (PCR), and real-time detection device and system |
| CN107460109A (en) * | 2017-07-23 | 2017-12-12 | 新疆昆泰锐生物技术有限公司 | A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument |
| CN108107024A (en) * | 2016-11-25 | 2018-06-01 | 苏州百源基因技术有限公司 | A kind of intelligence PCR instrument |
| CN109097455A (en) * | 2018-09-03 | 2018-12-28 | 中国科学院长春光学精密机械与物理研究所 | A kind of polymerase chain reaction system |
| CN111190447A (en) * | 2020-01-15 | 2020-05-22 | 广州大学 | Microfluid multi-temperature-zone temperature control system and method |
| CN113832020A (en) * | 2020-06-24 | 2021-12-24 | 傅宗民 | Optical module, thermal cycle module and PCR device suitable for PCR device |
| CN114054113A (en) * | 2022-01-16 | 2022-02-18 | 高分(北京)生物科技有限公司 | Heat-insulating reusable multifunctional cell counting imaging device without sample residue |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296351A (en) * | 2015-12-02 | 2016-02-03 | 北京大学 | Chip for polymerase chain reaction (PCR), and real-time detection device and system |
| CN108107024A (en) * | 2016-11-25 | 2018-06-01 | 苏州百源基因技术有限公司 | A kind of intelligence PCR instrument |
| CN107460109A (en) * | 2017-07-23 | 2017-12-12 | 新疆昆泰锐生物技术有限公司 | A kind of reaction system for bacterium colony PCR is prepared and sampling device and PCR instrument |
| CN109097455A (en) * | 2018-09-03 | 2018-12-28 | 中国科学院长春光学精密机械与物理研究所 | A kind of polymerase chain reaction system |
| CN109097455B (en) * | 2018-09-03 | 2022-11-11 | 中国科学院长春光学精密机械与物理研究所 | Polymerase chain reaction system |
| CN111190447A (en) * | 2020-01-15 | 2020-05-22 | 广州大学 | Microfluid multi-temperature-zone temperature control system and method |
| CN113832020A (en) * | 2020-06-24 | 2021-12-24 | 傅宗民 | Optical module, thermal cycle module and PCR device suitable for PCR device |
| CN114054113A (en) * | 2022-01-16 | 2022-02-18 | 高分(北京)生物科技有限公司 | Heat-insulating reusable multifunctional cell counting imaging device without sample residue |
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Application publication date: 20111019 |